To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production. We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 +/- 0.2 and 6.9 +/- 0.3 mM l(-1) respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system. A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.